Mass spectrometric quantitation of AGEs and enzymatic crosslinks in human cancellous bone

Abstract

Advanced glycation end-products (AGEs) deteriorate bone strength. Among over 40 species identified in vivo, AGEs other than pentosidine were roughly estimated as total fluorescent AGEs (tfAGEs) due to technical difficulties. Using LC-QqTOF-MS, we established a system that enabled the quantitation of five AGEs (CML, CEL, MG-H1, CMA and pentosidine) as well as two mature and three immature enzymatic crosslinks. Human bone samples were collected from 149 patients who underwent total knee arthroplasty. Their clinical parameters were collected to investigate parameters that may be predictive of AGE accumulation. All the analytes were quantitated and showed significant linearity with high sensitivity and precision. The results showed that MG-H1 was the most abundant AGE, whereas pentosidine was 1/200-1/20-fold less abundant than the other four AGEs. The AGEs were significantly and strongly correlated with pentosidine, while showing moderate correlation with tfAGEs. Interestingly, multiple linear regression analysis revealed that gender contributed most to the accumulation of all the AGEs, followed by age, tartrate-resistant acid phosphatase-5b and HbA1c. Furthermore, the AGEs were negatively correlated with immature crosslinks. Mass spectrometric quantitation of AGEs and enzymatic crosslinks is crucial to a better understanding of ageing- and disease-related deterioration of bone strength.

Figure 1

The pathways of AGEs formation.

Figure 2

AGEs analyzed in this study. (a) CML, N^ε-(carboxymethyl)lysine; (b) CEL, N^ε-(carboxyethyl)lysine; (c) MG-H1, N^δ-(5-hydro-5-methyl-4-imidazolon-2-yl)-ornithine 1; (d) CMA, N^ω-(carboxymethyl)arginine; (e) pentosidine.

Figure 3

Typical extracted ion chromatographic peaks of AGEs and enzymatic crosslinks (theoretical m/z ± 0.005). (a) CML, N^ε-(carboxymethyl)lysine; (b) CEL, N^ε-(carboxyethyl)lysine; (c) MG-H1, N^δ-(5-hydro-5-methyl-4-imidazolon-2-yl)-ornithine 1; (d) CMA, N^ω-(carboxymethyl)arginine; (e) pentosidine; (f) DHLNL, dihydroxylysinonorleucine; (g) HLNL, hydroxylysinonorleucine; (h) LNL, lysinonorleucine; (i) PYD, pyridinoline; (j) DPD, deoxypyridinoline.

Figure 4

AGEs and enzymatic crosslinks in human cancellous bone from lateral tibia plateau. The filled bar and error bar reflect mean value and standard deviation. Abbreviations: Hyp, hydroxyproline; CML, N^ε-(carboxymethyl)lysine; CEL, N^ε-(carboxyethyl)lysine; MG-H1, N^δ-(5-hydro-5-methyl-4-imidazolon-2-yl)-ornithine 1; CMA, N^ω-(carboxymethyl)arginine; DHLNL, dihydroxylysinonorleucine; HLNL, hydroxylysinonorleucine; LNL, lysinonorleucine; PYD, pyridinoline; DPD, deoxypyridinoline.